Tolerogenic dendritic cells, method for their production and uses therof

ABSTRACT

The present invention relates to a tolerogenic dendritic cell population (Tr-DC) capable of generating a population of T cells having regulatory activity, method of production and uses thereof. Furthermore, soluble HLA-G promotes the differentiation of a population of T cells with regulatory activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 12/300,764, filed Nov. 13, 2008, which is a 371 of PCTApplication Serial No. PCT/EP2007/002896, filed Mar. 30, 2007, which inturn claims the benefit of U.S. Provisional Application Ser. No.60/799,975, filed May 12, 2006, the contents of each of which areincorporated herein by reference in its entirety.

BACKGROUND OF INVENTION

Hematopoietic stem cell (HSC) transplantation is increasingly used forthe treatment of a number of malignant and non-malignant disorders ofboth hematopoietic and non-hematopoietic origin. However, rejectionresponses mediated by the immune system of the donor against therecipient, termed graft versus host disease (GvHD) remains a major causeof morbility. Organ transplantation is the best available establishedtechnique for the treatment of end stage failure of most essentialorgans (liver, heart, and lungs), but allograft rejection mediated bythe host is a major hurdle to long-term graft survival. A panel ofimmunosuppressive drugs is now available to prevent acute GvHD andallograft rejection including steroids, cyclosporin, metotrexate,cyclophosphamide, anti-thymocyte globulin, and anti-CD3 mAb. While theseagents have significantly improved graft outcomes, their use have beenassociated with numerous and rather significant toxicities. Moreover,continuous drug administration leads to a sustained state ofimmunosuppression with consequent high risk of infections. All theseeffects are linked to the non-selective mode of action of theimmunosuppressive drugs.

A valid alternative to immunosuppressive regimens for prevention of GvHDand of allograft rejection is the induction of tolerance to thealloantigens expressed by the recipient or the graft. This tolerancestrategy should selectively target only a small fraction of potentiallyalloreactive T cells and leave the rest of the immune system intact.

In autoimmune diseases, undesired immune response to self-antigens leadto destruction of peripheral tissues. Treatments of autoimmune diseasesare currently based on modulation of inflammation and non-specificimmunosuppression. Similarly to the prevention of allograft rejectionand GvHD, this approach is frequently not effective long-term due to theside effects of immunosuppression including infections and cancer, andhigh risk of disease relapse once the drug is withdrawn. An alternativestrategy is based on the induction of specific immune tolerance with theultimate goal to down-regulate the pathogenic immune response toself-antigens and to keep intact the mechanisms of host defense.

In chronic inflammatory diseases and in allergies an altered immuneresponse to pathogenic and non-pathogenic antigens occurs. This may bedue to an unbalance between effector and regulatory immune responses.Conventional anti-inflammatory or immunosuppressive therapies areinsufficient to restore this balance. Moreover, the benefit of thesetherapies is not long-lasting after drugs withdrawn. The induction ofantigen-specific tolerance mechanisms able to suppress undesiredresponses would represent a major advantage. Indeed, IL-10-producing Tcells with regulatory properties, which are specific for differentnon-pathogenic antigens have been isolated in healthy donors.

In addition to central tolerance which occurs during T-cell ontogeny inthe thymus and is mediated by clonal deletion of self-reactive T cells,peripheral T-cell tolerance is operational throughout life and isdesigned to control responses towards self antigens and foreign antigenswhich are not harmful. Peripheral T-cell tolerance can be induced andmaintained by a variety of mechanisms, including deletion, induction ofT-cell hypo-responsiveness, and differentiation of T regulatory (Tr)cells. Tr cells include a wide variety of cells with a unique capacityto inhibit effector T-cell responses. Although T cells with suppressiveactivity exist in all T-cell subsets, the best characterized arecomprised in the CD4⁺ T population. The two most relevant classes of Trcells described within the CD4⁺ subset to date are: T regulatory type 1(Tr1) cells (1) and CD4⁺CD25⁺ Tr cells (2). These two Tr cell subsetsdiffer in a number of important biological features, including theirspecific cytokine secretion profile, cellular markers, ability todifferentiate in response to Ag specific stimuli, and dependency oncytokines versus cell-cell contact mechanisms for mediating suppressiveactivity.

IL-10 and Type 1 T Regulatory (Tr1) Cells.

IL-10 plays a central role in controlling inflammatory processes,suppressing T cell responses, and maintaining immunological tolerance(reviewed in (3)). IL-10 inhibits IFN-γ and IL-2 production by T cells(4). It has anti-inflammatory effects inhibiting production ofpro-inflammatory cytokines, such as TNF-α, IL-1, and IL-6, andchemokines, such as IL-8 and MIP1α, produced by activatedantigen-presenting cells (APC), neutrophils, eosinophils, and mastcells. Furthermore, IL-10 down-regulates the expression of MHC class II,costimulatory, and adhesion molecules (5-7) on APC, and modulates theirstimulatory capacity (8). Importantly, IL-10 is crucial for thedifferentiation of adaptive type 1 T regulatory (Tr1) cells (1). Tr1cells are characterized by a unique cytokine secretion profile, upon TCRactivation they secrete high levels of IL-10, significant amounts ofIL-5, TGF-β and low levels of IFN-γ, and IL-2 but not IL-4 (1).Ag-specific murine Tr1 cells can be indeed differentiated in vitro byrepetitive TCR stimulation in the presence of high doses of IL-10 (1).Furthermore, treatment of mixed lymphocyte reaction (MLR) cultures withIL-10 (9) (and TGF-β in the mouse (10)) results in T-cell anergy.Importantly, allo-reactive Tr1 cell clones from healthy individuals havebeen originally isolated by limiting dilution of in vitroIL-10-anergized CD4⁺ T cells (1).

The first suggestion that human Tr1 cells are involved in maintainingperipheral tolerance in vivo came from studies in severe combinedimmunodeficient (SCID) patients successfully transplanted withHLA-mismatched allogenic stem cells. In the absence of immunosuppressivetherapy, these patients do not develop GvHD. Interestingly, high levelsof IL-10 are detected in the plasma of these patients and a significantproportion of donor-derived T cells, which are specific for the host HLAantigens and produce high levels of IL-10, can be isolated in vitro(11). Importantly, IL-10-anergized cells preserve their ability toproliferate in response to nominal antigens, such as Tetanus Toxoid andCandida Albicans, indicating that IL-10 induces an Ag-specific anergy(Bacchetta unpublished data). In a preclinical model of bone marrowtransplantation, transfer of donor CD4⁺ T cells anergized ex-vivo byhost APC in the presence of IL-10 and TGF-β results in a markedlydecreased GvHD in MHC class II mismatched recipients (10, 12). Thesedata offer a strong rationale for the development of a clinical protocolusing co-transfer of ex-vivo IL-10-anergized cells of donor origin inpatients undergoing haplo-identical HSC transplantation.

Tolerogenic Dendritic Cells (DC)

DC are highly specialized APC that classically initiate Ag-specificimmune responses upon infection (13). This process involves the terminalmaturation of DC, typically induced by agents associated with microbialinfection. It is now clear that DC can be not only immunogenic but alsotolerogenic. In steady state DC remains immature DC and can inducetolerance via deletion of Ag-specific effector T cells and/ordifferentiation of Tr cells (14-18). Repetitive stimulation of naïvecord blood CD4⁺ T cells with allogeneic immature DC results in thedifferentiation of IL-10-producing Tr cells (19), which suppress T-cellresponses via a cell-contact dependent mechanism. The authors recentlyreported that peripheral blood naïve CD4⁺ T cells stimulated withallogeneic immature DC become increasingly hypo-responsive tore-activation with mature DC and after three rounds of stimulation withimmature DC, they are profoundly anergic and acquire regulatoryfunction. These T cells are phenotypically and functionally similar toTr1 cells since they secrete high levels of IL-10 and TGF-β, suppressT-cell responses via an IL-10- and TGF-β-dependent mechanism, and theirinduction can be blocked by anti-IL10 mAb (20). Not only immature DC butalso specialized subsets of tolerogenic DC can drive the differentiationof Tr cells. Maturation and function of DC can be regulated at differentlevels (21). Both pharmacological and biological agents have been showncapable of inducing tolerogenic DC (22). Several biological agentsincluding IL-10 (23, 24), TGF-β (25), IFN-α (26, 27), and TNF-α (28) caninduce Tr cells. The presence of IL-10 during maturation of DC generatetolerogenic DC (23, 24), which express low levels of costimulatorymolecules and MHC class II (24), display low stimulatory capacity (3,29), and induce antigen-specific T cells anergy in both CD4⁺ and CD8⁺ Tcells (23, 24).

It has been already described that IL-10 during DC differentiationresults in a population of macrophage-like cells with low stimulatorycapacity but mature phenotype (8, 30). Herein, we demonstrated thatIL-10 treatment induces the differentiation of a unique subset of DC(Tr-DC) characterized by the expression of CD14, CD11c, CD11b, CD83,CD80, CD86, CD71 and HLA-DR, but not CD1a. Tr-DC expressimmunoglobulin-like transcript (ILT-) 2, ILT-3, ILT-4, and the nonclassical MCH class I molecule HLA-G. Tr-DC secrete significantly higherlevels of IL-10 compared to immature DC, whereas the amounts of IL-12are comparable to those produced by immature DC. Interestingly,IL-10/IL-12 ratio is maintained upon activation with LPS and IFN-γ.Tr-DC display lower stimulatory capacity compared to immature DC, and,importantly, induce Tr1 cells. Thus, IL-10 promote the differentiationof a new subset of tolerogenic DC which can be used to generate anergicTr1 cells with limited in vitro manipulation and suitable for potentialclinical use to restore peripheral tolerance.

Induction of T cell anergy by IL-10-treated DC has been suggested byZheng et al. (2004). The authors have generated immature DC by cultureof adherent cells with IL-4 and GM-CSF treatment. The immature DCobtained after 7 days are then washed and cultured with IL-10 foradditional 2 days. The resulting IL-10-treated immature DC present aphenotype very different from the one of the Tr-DC obtained in thepresent invention. Indeed, the cells obtained in Zheng et al. are CD83negative, CD86 low and HLA-DR low.

The protocol proposed by Levings et al. (2005) leads to the induction ofTr1 cells by repetitively stimulation of CD4⁺ T cells using immature DC,which are different from the Tr-DC generated in the present invention.

The international patent application WO2004/087899 discloses a methodfor obtaining Tr1 cells from T cells by means of specialized DC. DC areobtained from CD34⁺ cells in presence of IL-4, GM-CSF and IL-10.However, by contrast with the Tr1 DC of the present invention, theresulting DC express low level of CD11c, HLA-DR, CD80 and CD86, and areCD14 negative.

The international patent application WO03/000199 provides compositionswhich comprise at least two of a CD4⁺CD25⁺ T cell, IL-10, a CD8⁺CD28⁻cell and a vitamin D3 analog. This application also discloses a methodfor generating a tolerogenic antigen-presenting cell, which comprisescontacting the cell with an effective amount of IL-10, a CD4⁺CD25⁺ Tcell and/or a vitamin D3 analog. A method for increasing the expressionof ILT3 and/or ILT4 by an antigen-presenting cell which comprisescontacting the cell with an effective amount of IL-10, a CD4⁺CD25⁺ celland/or a vitamin D3 analog and methods for inhibiting the onset of ortreating the rejection of an antigenic substance and inhibiting theonset of or treating an autoimmune disease in a subject are provided.

The U.S. Pat. No. 6,277,635 describes IL-10 for producing a populationof cells which are capable of inhibiting or suppressing reactions toalloantigens, for example in graft-versus-host disease or tissuerejection. IL-10 for reducing responses in mixed lymphocyte response(MLR) is also described. Exogenous or induced endogenous IL-10 may beused for the inhibition or suppression of the reactions to alloantigens.The Tr-DC method of the present invention differs from the IL-10protocol to anergize T cells in vitro as follow:

-   -   Anergy by Tr-DC can be induced in all the individuals.    -   Anergic T cells induced by Tr-DC are more stable compared to        those obtained with IL-10.    -   T-cell cultures obtained with Tr-DC display higher cell recovery        compared to those obtained with IL-10.    -   IL-10 and Tr-DC are comparable in inducing T-cell anergy in        haplo-identical pairs. Importantly, in haplo-identical pairs in        which IL-10 does not induce anergy, Tr-DC do.    -   In HLA-matched un-related (MUD) pairs the use of DC is required        to stimulate host-specific T-cell responses, therefore Tr-DC are        necessary for T-cell anergy induction.    -   Lower number of cells from both recipient and donor are required        for the in vitro manipulation to generate anergized T cells with        the Tr-DC of the present invention.

The United States patent application 20070009497 relates toculture-expanded T suppressor cells and their use in modulating immuneresponses. This invention provides methods of producing culture-expandedT suppressor cells, which are antigen specific, and their use inmodulating complex autoimmune diseases. In particular a method forproducing an isolated, culture-expanded T suppressor cell population,comprising: (a) contacting CD25⁺CD4⁺ T cells with DC and an antigenicpeptide, an antigenic protein, or a derivative thereof, or an agent thatcross-links a T cell receptor on said T cells in a culture, for a periodof time resulting in antigen-specific CD25⁺CD4⁺ T cell expansion; and(b) isolating the expanded CD25⁺CD4⁺ T cells obtained in (a), therebyproducing an isolated, culture-expanded T suppressor cell population isprovided. The DC population describes in this application display verydifferent characteristics than the Tr-DC population of the presentinvention.

The International patent application WO03102162 relates to tolerogenicDC and methods for enriching for these cells in tissue preparations andusing the cells for preventing or minimizing transplant rejection or fortreating or preventing an autoimmune disease. A human tolerogenic DChaving surface antigens DEC205 and B220, but not CD19 is described.

HLA-G and Immunomodulatory Properties

HLA-G, a non-classical MHC class I molecules, is a low polymorphicmolecule. Compared with the classical class I genes, the mostpolymorphic genes in the human genome, HLA-G has relatively littlepolymorphism in its coding region (31). The HLA-G gene has eight exonsencoding a signal peptide (exon 1), the α1, α2, and α3 domains (exons 2,3, and 4, respectively), the transmembrane domain (exon 5), and theintracellular domain (exons 6 and 7), similar to other class I genes.However, a premature stop codon in exon 6 results in a truncatedcytoplasmic tail that reveals a cryptic retrieval motif (32). Thisresults in the slow turnover and prolonged expression of HLA-G at thecell surface. HLA-G encodes multiple isoforms as a result of alternativesplicing. The full-length isoform HLA-G1 is structurally similar toother class I genes, except for the truncated cytoplasmic tail. The G2isoform results from the removal of exon 3 and homodimerizes to form anHLA class II-like structure (33). HLA-G1 and HLA-G2 isoforms can be alsoexpressed as soluble proteins (HLA-G5 and -G6, respectively) due to theinclusion of intron 4 sequences in the mature mRNA, resulting insecreted proteins with an additional 21 amino acids (encoded by intron 4sequences) following the α3 domain (34). HLA-G3 results from the removalof exons 3 and 4. Additional isoforms are HLA-G4 and -G7.

HLA-G has been extensively studied in pregnancy and it is known to bethe major contributor to induction and maintenance of foetal-maternaltolerance (31, 35). HLA-G inhibits cytolytic activities of both NK andCTL (36), and allo-specific T-cell proliferation (37, 38). A positivecorrelation between allograft acceptance and HLA-G expression on bothgraft cells (39, 40) and T cells (38) has been reported (41), indicatinga role of HLA-G in modulating allo-responses. In addition, HLA-G acts asa negative regulator of tumor immune responses through severalmechanisms including, inhibition of angiogenesis, prevention of antigenrecognition and T-cell migration, and suppression of T and NK cytolyticeffects (42). Antigen-presenting cells expressing HLA-G1 are poorstimulators and are able to promote the induction of anergic/suppressorCD4⁺ T cells (43). Moreover, HLA-G binds to the inhibitory moleculesimmunoglobulin-like transcript (ILT)-2 and ILT-4 expressed on DC (39,44). It has been shown that engagement of ILT-4 by HLA-G prevents theup-regulation of costimulatory molecules, inhibits DC maturation (45),and promotes the differentiation of anergic/suppressor CD4⁺ T cells(46). The authors demonstrated that soluble HLA-G alone or incombination with IL-10 promotes the differentiation of a population ofCD4⁺ T cells with low proliferative capacity and suppressor functions.Soluble HLA-G-induced Tr cells produce TGF-β, intermediate levels ofIL-10 and IFN-γ, but low levels of IL-2, and IL-4, express high levelsof granzyme B, CTLA4, CD25, but not FOXP3. Thus soluble HLA-G is a newimmunomodulatory compound able to promote the differentiation of apopulation of CD4⁺ T cells with regulatory activity.

SUMMARY OF INVENTION

In the present invention the following nomenclature was used:

Tr1 for Type 1 T regulatory, iDC for immature dendritic cells, Tr-DC fordendritic cells generated in the presence of exogenous IL-10. Tr-Dc maybe also called Tr1-DC, DC-10, and IL-10 DC. mDC for mature dendriticcells, T(iDC) for T cell lines generated by stimulating naïve CD4⁺ Tcells or PBMC with allogeneic immature DC, T(Tr-DC) for T cell linesgenerated by stimulating naïve CD4⁺ T cells or PBMC with Tr-DC, T(mDC)for T cell lines generated by stimulating naïve CD4⁺ T cells or PBMCwith mature DC, T(MLR) for T cell lines generated by stimulating PBMCwith allogenic CD3 depleted cells, T(MLR/IL-10) for T cell linesgenerated by stimulating PBMC with allogenic CD3 depleted cells in thepresence of exogenous IL-10, Th0 for T cell lines differentiated invitro in the presence of exogenous IL-2, Tg for T cell linesdifferentiated in vitro in the presence of soluble HLA-G, Tg10 for Tcell lines differentiated in vitro in the presence of exogenous IL-10and soluble HLA-G.

The present invention relates to a method to generate T cells havingregulatory activity in particular, Tr1 cells using a unique populationof dendritic cells named Tr-DC. Furthermore, the ability of solubleHLA-G to promote the differentiation of regulatory T cells is disclosed.The potential to generate T cells having regulatory activity to be usedas cellular therapy in the clinical context of allogeneic HSCtransplantation, organ transplantation, autoimmune diseases, chronicinflammatory diseases, allergies, and asthma with limited in vitromanipulation is valuable.

It is therefore an object of the invention a tolerogenic dendritic cellpopulation (Tr-DC) having the following marker phenotype: CD14⁺, CD11c⁺,CD11b⁺, and CD1a⁻. Preferably, the tolerogenic dendritic cell population(Tr-DC) is further CD83⁺, CD80⁺, CD86⁺, HLA-DR⁺, CD71⁺. More preferablythe tolerogenic dendritic cell population is further ILT-2⁺ and/orILT-3⁺ and/or ILT-4⁺ and/or HLA-G⁺.

Even more preferably, the tolerogenic dendritic cell population (Tr-DC)is capable to generate a population of T cells having regulatoryactivity. Preferably the population of T cells having regulatoryactivity is a population of Tr1 cells.

It is an object of the invention an in vitro method for generating apopulation of tolerogenic dendritic cells (Tr-DC) as defined abovecomprising the steps of:

-   -   a) collecting PBMCs from a subject;    -   b) isolating adherent cells from collected PBMCs;    -   c) exposing said isolated adherent cells under appropriate        culture conditions to an effective amount of GM-CSF, IL-4 and        IL-10 or functional derivatives thereof.

Preferably said adherent cells are mainly CD14⁺ monocytes. Preferablythe step of isolating adherent cells and exposing said isolated adherentcells under appropriate culture conditions, is performed in the presenceof fetal calf serum (FCS) or of human serum (HS). Preferably, theeffective amount of GM-CSF is between 1-1000 ng/ml. Preferably, theeffective amount of IL-4 is between 1-1000 ng/ml. Preferably, theeffective amount of IL-10 is between 1-1000 ng/ml.

It is a further object of the invention a method for isolating apopulation of tolerogenic dendritic cells (Tr-DC) as described abovecomprising the steps of:

-   -   a) collecting a sample from a subject;    -   b) isolating the sample cells with at least one of markers        included in the group of: CD14, CD11c, CD11b, CD83, CD80, CD86,        HLA-DR, CD71, ILT-2, ILT-3, ILT-4 or HLA-G.

Preferably, the sample is a blood, a spleen or a lymph node sample.

It is an object of the invention, the use of the population oftolerogenic dendritic cells Tr-DC as described above for generating apopulation of T cells having regulatory activity. Preferably, thepopulation of T cells having regulatory activity is a population of Tr1cells.

It is a further object of the invention an in vitro method forgenerating a population of T cells having regulatory activity comprisingthe steps of:

-   -   a) irradiating the Tr-DC cell population described above;    -   b) isolating PBMCs from a subject;    -   d) stimulating said isolated PBMCs in appropriate culture        conditions with an effective amount of said irradiated Tr-DC        cell population.

Preferably, in the in vitro method, the population of T cells havingregulatory activity is a population of Tr1 cells.

It is another object of the invention, a population of Tr1 cellsobtainable by the method described above being:

a) anergic;

b) T cell response suppressive;

c) DC response suppressive; and

d) having the following marker phenotype: IL-10⁺⁺, TGF-β⁺, IL-4⁻ andIFN-γ and IL-2 negative to low.

It is another object of the invention, the use of the population of Tcells having regulatory activity obtainable according to the methodabove to induce or restore immune tolerance in a subject.

It is a further object of the invention, the use of the population of Tcells having regulatory activity obtainable according to the methoddescribed above for the preparation of a medicament for the preventionand/or treatment of graft versus host disease, and/or of organrejection, and/or of autoimmune diseases, and/or of allergies, and/or ofasthma, and/or of chronic inflammatory diseases. Preferably, theautoimmune diseases are comprised in the group of: type 1 diabetesmellitus, autoimmune enteropathy, rheumatoid arthritis, systemic lupuserythematosus, multiple sclerosis or psoriasis. Preferably, the chronicinflammatory diseases are comprised in the group of: inflammatory boweldisease, Crohn's disease or vasculitis. More preferably allergiescomprise atopic dermatitis.

It is a further object of the invention the use of the population of Tcells having regulatory activity obtainable according to the methodabove for the preparation of a medicament for the prevention and/ortreatment of immune responses induced by gene therapy products.

Another object of the invention is the use of the population of T cellshaving regulatory activity obtainable according to the method above forthe treatment of genetic autoimmune diseases comprised in the group of:immune dysfunction, Polyendocrinopathy Enteropathy X-linked (IPEX)syndrome, Autoimmune Polyendocrinopathy-Candidiasis-Ectodermal Dystrophy(APECED) syndrome, and OMENN's syndrome.

Preferably, the population of T cells having regulatory activity is apopulation of Tr1 cells. Another object of the invention is the use ofthe tolerogenic dendritic cell population (Tr-DC) as described above toinduce or restore immune tolerance in a subject.

It is also an object of the invention the use of the tolerogenicdendritic cell population (Tr-DC) for the preparation of a medicamentfor the prevention and/or treatment of graft versus host disease, and/orof organ rejection, and/or of autoimmune diseases, and/or of allergies,and/or of asthma, and/or of chronic inflammatory diseases. Preferablythe autoimmune diseases are comprised in the group of: type 1 diabetesmellitus, autoimmune entheropathy, rheumatoid arthritis, systemic lupuserythematosus, multiple sclerosis or psoriasis. Preferably the chronicinflammatory diseases are comprised in the group of: inflammatory boweldisease, Chron's disease or vasculitis. Preferably, allergies compriseatopic dermatitis.

It is a further object of the invention, the use of the tolerogenicdendritic cell population (Tr-DC) as described above for the preparationof a medicament for the prevention and/or treatment of immune responsesinduced by gene therapy products.

It is another object of the invention, the use of the tolerogenicdendritic cell population (Tr-DC) as described above for the treatmentof genetic autoimmune diseases comprised in the group of: immunedysfunction, Polyendocrinopathy Enteropathy X-linked (IPEX) syndrome,Autoimmune Polyendocrinopathy-Candidiasis-Ectodermal Dystrophy (APECED)syndrome, and OMENN's syndrome.

It is an object of the invention the use of HLA-G as a tolerogenicbiomarker of Tr-DC.

It is a further object of the invention the use of soluble HLA-G togenerate a population of T cells having regulatory activity.

It is another object of the invention the use of soluble HLA-G to induceor restore immune tolerance in a subject and the use of soluble HLA-Gfor the preparation of a medicament for the prevention and/or treatmentof graft versus host disease, and/or of organ rejection, and/or ofautoimmune diseases, and/or of allergies, and/or of asthma, and/or ofchronic inflammatory diseases.

Preferably soluble HLA-G is soluble HLA-G1 and/or HLA-G5.

In the methods of the invention, the subject from whom Tr-DC aregenerated may be different from the subject from whom PBMCs areisolated. The subject from whom PBMCs are isolated may be a recipient inthe case of hematopoietic stem cell transplantation, a donor in the caseof organ transplantation, or a self in the case of autoimmunity,allergies, asthma, and chronic inflammatory diseases. The methods of thepresent invention are independent on the degree of HLA disparitiesbetween the Tr-DC and PBMCs cells used.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be now described by means of non limiting examplesreferring to the following figures:

FIG. 1. Tr-DC: morphology and phenotype. Monocyte-derived DC weredifferentiated in IL-4 and GM-CSF in the presence of IL-10 (Tr-DC) for 7days, or in IL-4 and GM-CSF for 5 days and cultured for additional 2days with (mature DC) or without (immature DC) LPS. A. Morphology of DCwas evaluated by microscopy. B. Expression of CD1a, CD14, CD83, HLA-DR,CD11c, CD11b, CD71, CD80, and CD86 was evaluated by FACS analysis. Arepresentative donor out of twenty tested in independent experiments ispresented. C-D. Tr-DC produce high levels of IL-10 but low amounts ofIL-12. Immature (iDC), DC differentiated with IL-10 (Tr-DC), and matureDC (mDC) were cultured (C) alone or (D) activated with IFN-γ (50 ng/ml)and LPS (200 ng/ml). Culture supernatants were collected 48 h afterculture, and levels of secreted IL-12 and IL-10 were determined byELISA. The average ±SEM amounts detected in five independent experimentsare presented. *P≤0.05 and **P≤0.005 as indicated.

FIG. 2. Expression of mRNA for IL-10 and IL-12 in iDC, Tr-DC, and mDC(A) left inactivated or (B) activated with IFN-γ (50 ng/ml) and LPS (200ng/ml), were compared for. Relative levels of IL-10 and IL-12 expressionwere determined by quantitative RT-PCR. The amounts of IL-10 and IL-12mRNA are expressed as relative to non-activated PBMC (which were givenan arbitrary value of 1). The average ±SEM amounts detected in sixindependent experiments are presented. *P<0.05, and **P<0.005 whencompared to iDC.

FIG. 3. Tr-DC display low stimulatory capacity. A. Naïve CD4⁺ T cellswere cultured with allogeneic immature (iDC), DC differentiated in thepresence of IL-10 (Tr-DC), and mature DC (mDC) at the ratio of 10:1.Proliferate responses were evaluated 4 days after culture by[³H]-thymidine incorporation for an additional 16 h. B. In parallel,supernatants were collected after 48 h and IFN-γ analyzed by ELISA. C.Activated Tr-DC maintain low stimulatory capacity. Allogeneic iDC,Tr-DC, and mDC, activated with IFN-γ (50 ng/ml) and LPS (200 ng/ml) for48 h, were cultured with naïve CD4⁺ T cells at the ratio of 1:10.Proliferative responses were evaluated 4 days after culture by[³H]-thymidine incorporation for an additional 16 h. Results of onerepresentative experiment of twenty-four (A), four (B), and eight (C)independent experiments performed are shown. Numbers represent the % ofinhibition of proliferation of T cells primed with iDC or Tr-DC comparedto proliferation of T cells stimulated with mDC (A, C), the % ofinhibition of IFN-γ production by T cells primed with iDC or Tr-DCcompared to that obtained in T cells stimulated with mDC (B). **P≤0.005when naïve CD4⁺ T cells primed with Tr-DC were compared to naïve CD4⁺ Tcells primed with iDC.

FIG. 4. Tr-DC induce T-cell anergy. A. To generate anergic T cells,naïve CD4⁺ T cells were stimulated with allogeneic iDC [T(iDC)], Tr-DC[T(Tr-DC)], or mDC [T(mDC)] for one or two rounds of stimulation. Afterone round (A, B) and two rounds (C) of stimulation, T(iDC), T(Tr-DC),and T(mDC) cell lines were tested for their ability to proliferate inresponse to mDC from the same allogeneic donor. Proliferative responseswere evaluated 2 days after culture by [³H]-thymidine incorporation foran additional 16 h. B. In parallel, supernatants were collected after 48h and analyzed by ELISA to determine levels of IFN-γ. Results of onerepresentative experiment of twenty-four (A), three (B), and eight (C)independent experiments performed are shown. Numbers represent the % ofanergy of T(iDC) or T(Tr-DC) cell lines compared to T(mDC) cell lines.**P≤0.005 when T(Tr-DC) cell lines were compared to T(iDC) cell lines.

FIG. 5. Tr-DC induce Tr1 cells. Naïve CD4⁺ T cells were stimulated withallogeneic iDC [T(iDC)], Tr-DC [T(Tr-DC)], or mDC [T(mDC)] one or tworounds of stimulation. At the end of one (A) or two (B) rounds ofstimulation T cell lines were restimulated with immobilized anti-CD3 mAb(10 μg/ml) and TPA (1 ng/ml), and cytokine production was determined byintracytoplasmic staining and cytofluorometric analysis, as described inMaterials and Methods. One representative experiment out of nine (A) orthree (B) is presented. C. At the end of each round of stimulations withimmature [T(iDC)], Tr-DC [T(Tr-DC)] and mature DC [T(mDC)], T-cell lineswere activated with mDC and supernatants were collected after 72 h ofculture. Levels of for TGF-β were determined by ELISA. The average ±SEMamounts detected in five independent experiments are presented.

FIG. 6. Tr-DC induce Tr1 cells. Naïve CD4⁺ T cells were stimulated withallogeneic iDC [T(iDC)], Tr-DC [T(Tr-DC)], or mDC [T(mDC)] for 14 days(one round of stimulation). After stimulation, T-cell lines wereactivated with immobilized anti-CD3 mAb and TPA, and cytokine productionwas determined by intracytoplasmic staining and cytofluorometricanalysis. Percentages of IFN-γ (A), IL-2 (B), IL-4 (C), and IL-10(D)-producing cells in T(iDC), T(Tr-DC), and T(mDC) cell lines generatedfrom each of the nine donors tested are presented.

***P≤0.001 when T(Tr-DC) cell lines were compared to T(iDC) cell lines.

FIG. 7. Phenotype of T cells generated with Tr-DC (A), T(iDC), T(Tr-DC),and T(mDC) cell lines was analyzed 14 days after culture for theexpression of the indicated markers (B). Results from one representativedonor out of nine tested are presented.

FIG. 8. Tr-DC are more powerful than immature DC to generate Tr1 cells.Naive CD4⁺ T cells were stimulated with allogeneic immature [T(iDC)],[T(Tr-DC)] and mature DC [T(mDC)] for one or two rounds of stimulation.A. After one round of stimulation, T-cell lines were tested for theirability to suppress responses of autologous CD4⁺ T cells activated withmDC (MLR). Naïve CD4⁺ T cells were stimulated with mDC alone (MLR) or inthe presence of T(iDC), T(Tr-DC), and T(mDC) cell lines at a 1:1 ratio.[³H]-thymidine was added after 3 days of culture for an additional 16 h.Results of one experiment representative of eight independentexperiments are shown. B. Suppression of IFN-γ production by CD4⁺ Tcells in response to mDC was measured in culture supernatants after 4days of culture. Results representative of three independent experimentsare shown. C-D. Kinetic of suppression by T(Tr-DC) cells. Naïve CD4⁺ Tcells were stimulated with allogeneic iDC [T(iDC)], Tr-DC [T(Tr-DC)], ormDC [T(mDC)] for one or two rounds of stimulation. After one (C) and two(E) rounds of stimulation, T-cell lines were tested for their ability tosuppress responses of autologous CD4⁺ T cells activated with mDC (MLR).Naïve CD4⁺ T cells were stimulated with mDC alone (MLR) or in thepresence of T(iDC), T(Tr-DC), and T(mDC) cell lines at a 1:1 ratio.[³H]-thymidine was added at day 2, 3, and 4 for an additional 16 h.Results of one experiment representative of eight independentexperiments are shown. D. After one round of stimulation, T cell lineswere tested for their ability to suppression of IFN-γ production by CD4⁺T cells in response to mDC. Results representative of three independentexperiments are shown.

FIG. 9 A-B. Role of IL-10 and TGF-β in suppression mediated by T(Tr-DC)cell lines. T(Tr-DC) cell lines were tested for their ability tosuppress IFN-γ production of CD4⁺ T cells in response to allogeneicmonocytes in the absence or presence of anti-IL-10R and anti-TGF-β mAbs.Suppression of IFN-γ production was measured in culture supernatants 2(B), 3 (B), and 4 (A, B) days after culture. Results are representativeof three independent experiments. C-D. Autocrine IL-10 is required forthe differentiation of T(Tr-DC) cells with regulatory activity. NaiveCD4⁺ T cells were stimulated with allogeneic Tr-DC in the presence ofanti-IL10R or control IgG mAbs. After activation, T cells were collectedand tested for their ability to suppress the response of autologous CD4⁺T cells activated with mDC (MLR). [³H]-thymidine was added at day 2 (D),3 (D), and 4 (C, D) for an additional 16 h. Results of one experimentrepresentative of three independent experiments are shown.

FIG. 10. Tr-DC express tolerogenic markers. Monocyte-derived DC weredifferentiated in IL-4 and GM-CSF in the presence of IL-10 (Tr-DC) for 7days, or in the absence of IL-10 for 5 days and activated for additional2 days with (mDC) or without (iDC) LPS. A. DC were analyzed by flowcytometry to determine levels of expression of ILT-2, ILT-3, ILT-4,HLA-G, and ICOS-L. B. Mean percentages of positive cells, set accordingto the isotype-matched controls, gated on CD11c⁺ cells (not shown), ±SDare shown. *P<0.01 when TR-DC were compared to iDC and mDC. C-G.Induction of Tr1 cells requires ILT-4/HLA-G interaction. Naïve CD4⁺ Tcells were stimulated with Tr-DC in presence of anti-ILT-4 [T(Tr-DCanti-ILT-4)] or control IgG [T(Tr-DC)] mAbs. As control, naïve CD4⁺ Tcells were stimulated with mDC [T(mDC)]. After stimulation, T cell lineswere collected and tested for their ability to proliferate in responseto mDC (C) and to suppress responses of autologous CD4⁺ T cellsactivated with mDC (MLR) (D). [³H]-thymidine was added after 2 days (C),and 4 days (D) of culture for an additional 16 h. Results arerepresentative of four independent experiments. E. IL-10 inducesup-regulation of HLA-G on naïve T cells. Naïve CD4⁺ T cells werestimulated with iDC, Tr-DC, and mDC for 48 hours in the presence ofcontrol IgG or anti-IL-10R mAbs. T cells were analyzed by flow cytometryto determine levels of expression of HLA-G. Percentages of CD4⁺HLA-G⁺ Tcells are shown. Red lines represent the mean percentages of CD4⁺HLA-G⁺T cells. F-G. Naïve CD4⁺ T cells were stimulated with Tr-DC in thepresence of anti-HLA-G [T(Tr-DC anti-HLA-G)] or control IgG [T(Tr-DC)]mAbs. As control, naïve CD4⁺ T cells were stimulated with mDC [T(mDC)].After stimulation, T cell lines were collected and tested for theirability to proliferate in response to mDC (F) and to suppress responsesof autologous CD4⁺ T cells activated with mDC (MLR) (G). [³H]-thymidinewas added after 2 days (F), and 4 days (G) of culture for an additional16 h. Results are representative of three independent experiments.

FIG. 11. Kinetic of suppression by T(Tr-DC) cells in the presence ofanti-ILT-4 or anti-HLA-G mAbs. A. Naïve CD4⁺ T cells were stimulatedwith Tr-DC in presence of anti-ILT-4 [T(Tr-DC+anti-ILT-4)] or controlIgG [T(Tr-DC)] mAbs. As control, naïve CD4⁺ T cells were stimulated withmDC [T(mDC)]. After stimulation, T cell lines were collected and testedfor their ability to suppress responses of autologous CD4⁺ T cellsactivated with mDC (MLR). [³H]-thymidine was added after 2, 3, and 4days of culture for an additional 16 h. Results are representative offour independent experiments. B. Naïve CD4⁺ T cells were stimulated withTr-DC in the presence of anti-HLA-G [T(Tr-DC+anti-HLA-G)] or control IgG[T(Tr-DC)] mAbs. As control, naïve CD4⁺ T cells were stimulated with mDC[T(mDC)]. After stimulation, T cell lines were collected and tested fortheir ability to suppress responses of autologous CD4⁺ T cells activatedwith mDC (MLR). [³H]-thymidine was added after 2, 3, and 4 days ofculture for an additional 16 h. Results are representative of threeindependent experiments.

FIG. 12. Lack of stimulation capacity of Tr-DC. PBMC were cultured withallogeneic cells differentiated with IL-10 (Tr-DC) and mature DC (mDC)at ratio 1:10. A. Proliferate responses were evaluated 4 days afterculture by [³H]-thymidine incorporation for an additional 16 h B. Inparallel, supernatants were collected after 48 hours and analyzed byELISA to determine levels of IFN-γ. Numbers represent the % ofinhibition of proliferation compared to that obtained with mDC.

FIG. 13. Tr-DC induce anergic T cell. Total PBMC were stimulated withallogeneic Tr-DC [T(Tr-DC)] and mature DC [T(mDC)] at 1:10 ratio for tendays. After culture, T-cell lines were tested for their ability toproliferate in response to mature allogeneic DC. Proliferative responseswere evaluated after 2 days of culture by [³H]-thymidine incorporationfor an additional 16 h. Numbers represent the % of anergy compared tomDC.

FIG. 14. Tr-DC induce anergic T cells in haplo-identical pairs. TotalPBMC were stimulated with haplo-identical Tr-DC [T (Tr-DC)] or mature DC[T(mDC)] at 1:10 ratio for ten days. After culture, T-cell lines weretested for their ability to proliferate in response to mature allogeneicDC. Proliferative responses were evaluated by thymidine incorporationafter 2 days of culture by [³H]-thymidine incorporation for anadditional 16 h. Numbers represent the % of anergy compared to mDC.

FIG. 15. Tr-DC induce anergic T cells in HLA-matched un-related (MUD)pairs. PBMC were stimulated with Tr-DC [T(Tr-DC)] or mature DC [T(mDC)]at 1:10 ratio for ten days. After culture, T-cell lines were tested fortheir ability to proliferate in response to mature allogeneic DC.Proliferative responses were evaluated by thymidine incorporation after2 days of culture by [³H]-thymidine incorporation for an additional 16h. Numbers represent the % of anergy compared to mDC.

FIG. 16. Tr-DC are equivalent to exogenous IL-10 to generate anergic Tcells in haplo-identical pairs. Total PBMC were stimulated with Tr-DC[T(Tr-DC)] or mature DC [T(mDC)] at 10:1 ratio (A) or with CD3-depletedcells in the absence [T(MLR)] or in the presence of exogenous IL-10[T(MLR/IL-10)] at 1:1 ratio for ten days (B). After culture, T-celllines were tested for their ability to proliferate in response to matureallogeneic DC. Proliferative responses were evaluated after 2 days ofculture by [³H]-thymidine incorporation for an additional 16 h. Numbersrepresent the % of anergy compared to mDC.

FIG. 17. Comparison between Tr-DC generated in medium containing FBS orHS. Monocyte-derived DC were differentiated in IL-4 and GM-CSF in thepresence of IL-10 (Tr-DC) for 7 days in medium containing FBS or HS. A.Expression levels of CD1a, CD14, CD83, HLA-DR, CD83, CD80, CD86, CD11cand CD11b were evaluated by FACS analysis. B. Total PBMC were stimulatedwith Tr-DC [T(Tr-DC)] or mature DC [T(mDC)] generated in mediumcontaining FCS or HS at 1:10 ratio for ten days. After culture, T-celllines were tested for their ability to proliferate in response to matureallogeneic DC. Proliferative responses were evaluated after 2 days ofculture by [³H]-thymidine incorporation for an additional 16 h. Numbersrepresent the % of anergy compared to mDC.

FIG. 18. Comparison between Tr-DC generated in flask and plate.Monocyte-derived DC were differentiated in IL-4 and GM-CSF in thepresence of IL-10 (Tr-DC) for 7 days in flask or plate. Total PBMC werestimulated with Tr-DC [T(Tr-DC)] or mature DC [T(mDC)] at 1:10 ratio forten days. After culture, T-cell lines were tested for their ability toproliferate in response to mature allogeneic DC. Proliferative responseswere evaluated after 2 days of culture by [³H]-thymidine incorporationfor an additional 16 h. Numbers represent the % of anergy compared tomDC.

FIG. 19. Tr-DC are present in peripheral blood and human spleen.Expression levels of CD11c, CD11b, CD14, CD1a, CD80, CD83, CD86, CD71,and HLA-DR in peripheral blood (A), and in human spleens (B) wereevaluated by FACS analysis. Analyses were performed on CD11b⁺CD11c⁺gated cells. Filled histograms represent staining with theisotype-matched control mAbs. A representative donor out of six (A) andfour (B) independent donors analyzed is presented. Percentages ofCD11c⁺CD11b⁺ cells expressing the indicated markers are indicated.

FIG. 20. Tr-DC present in peripheral blood and human spleens express thetolerogenic markers ILT-2, ILT-3, ILT-4, and HLA-G. Freshly isolatedcells from peripheral blood (A) and human spleens (B) were analyzed byflow cytometry to determine levels of expression of ILT-2, ILT-3, ILT-4,and HLA-G. Analyses were performed on CD11b⁺CD11c⁺ gated cells. Filledhistograms represent staining with the appropriate control mAbs. Datafrom one out of six (A), and four (B) independent donors analyzed arepresented. Percentages of CD11c⁺CD11b⁺ cells expressing the indicatedmarkers are indicated.

FIG. 21. Tr1 cells induction via the IL-10-dependent ILT-4/HLA-Gpathway. Tr-DC secrete high levels of IL-10 (1). During T-cell priming,IL-10 produced by Tr-DC inhibits T cell proliferation (2) and promotesthe up-regulation of HLA-G on allogeneic CD4⁺ T cells (3). IL-10up-regulates ILT-2, ILT-3, ILT-4, and HLA-G on DC (4). HLA-G express onT cells interacts with ILT-4 on Tr-DC (5) and enhances IL-10 secretion(6). Tr-DC-derived IL-10 promotes de novo differentiation of tolerogenicDC by inducing ILT-2, ILT-3, ILT-4, and HLA-G expression (7).Concomitantly, interaction between ILT-2/ILT-4 on TR-DC and HLA-G on Tcells, and HLA-G on Tr-DC and ILT-2 on T cells provides negative signalsto T cells with further inhibition of their proliferation and cytokineproduction (8). This effect promotes T-cell anergy when T cells arere-challenged with the same Ag (9) and Tr1 cell differentiation (10).Tr1 cells secrete IL-10, which contributes to amplify this tolerogeniccircuit (11).

FIG. 22. Cytokine production profile of T cell lines differentiated inthe presence of soluble HLA-G. Naïve CD4⁺ T cells were activated byanti-CD3 mAbs cross-linked on CD32+CD80+CD58+ L cells in the presence ofexogenous IL-2 (Th0), soluble HLA-G1 (30 ng/ml) (Tg), soluble HLA-G1 (30ng/ml) and exogenous IL-10 (10 ng/ml) (Tg10), or exogenous IL-10 (10ng/ml) and IFN-α (5 ng/ml) (Tr1). A. Following two rounds of identicalstimulation T cells were restimulated with immobilized anti-CD3 mAb (1μg/ml) and TPA (10 ng/ml), and cytokine production was determine byintracytoplasmic staining. Percentages set according to theisotype-matched controls (not shown), are presented. One representativeexperiment out of six independent experiments is shown. B. Following tworounds of identical stimulation T cells were restimulated with coatedanti-CD3 mAb (1 μg/ml) and soluble anti-CD28 (10 μg/ml). Culturesupernatants were collected after 24 h, 48 h, and 72 h. IL-2 (24 h),IFN-γ, IL-10, and TGF-β (48 h) levels were determined by ELISA. Meanlevels of cytokines collected in 5 experiments, ±StD are shown.

FIG. 23. Phenotype of T cell lines differentiated in the presence ofsoluble HLA-G. Naïve CD4⁺ T cells were activated by anti-CD3 mAbscross-linked on CD32+CD80+CD58+ L cells in the presence of exogenousIL-2 (Th0), soluble HLA-G1 (30 ng/ml) (Tg), soluble HLA-G1 (30 ng/ml)and exogenous IL-10 (10 ng/ml) (Tg10), or exogenous IL-10 (10 ng/ml) andIFN-α (5 ng/ml) (Tr1). Following two rounds of identical stimulation Tcells were analyzed for the expression of the indicated markers. The MFI(upper number) and the percentages (lower number) of positive cells, setaccording to the isotype-matched controls (not shown), are presented.Results from one representative experiment out of three (A), four (B),and six (C) performed are presented.

FIG. 24. T cells differentiated with soluble HLA-G alone or incombination with IL-10 are suppressor cells. Naïve CD4⁺ T cells wereactivated by anti-CD3 mAbs cross-linked on CD32+CD80+CD58+ L cells inthe presence of exogenous IL-2 (Th0) (A), soluble HLA-G1 (30 ng/ml) (Tg)(B), soluble HLA-G1 (30 ng/ml) and exogenous IL-10 (10 ng/ml) (Tg10)(C), or exogenous IL-10 (long/ml) and IFN-α (5 ng/ml) (Tr1) (D).Following two rounds of identical stimulation T cells were tested fortheir ability to suppress proliferation CSFE-labeled autologousCD4⁺CD45RO⁺ T cells were stimulated with coated anti-CD3 mAb (10 μg/ml)and soluble anti-CD28 (1 μg/ml) in the presence (open histograms) orabsence (closed histograms) of Th0, Tg, Tg10, and Tr1 at 1:1 ratio.After 6 days were analyzed by flow cytometry. Percentages of suppressionare presented. One representative experiment out of four independentexperiments.

FIG. 25. Tr1-DC are differentiated in vitro from CD14⁺ cells isolatedfrom PBMC in the presence of GM-CSF/IL-4/IL-10. After differentiation,Tr1-DC cells display the characteristics described in the middle upperbox and are cultured with PBMC. Anergic allo-antigen specific Tr1 cellswith suppressive activity are generated. These cells exhibit thecharacteristics described in the lower box and are used as cell-basedtherapy to restore peripheral tolerance.

DETAILED DESCRIPTION OF THE INVENTION

Material and Methods

Cell Preparations

Human peripheral blood was obtained from healthy donors in accordancewith local ethical committee approval. Peripheral blood mononuclearcells (PBMC) were separated by density gradient centrifugation overLymphoprep (Nycomed Amersham). Human spleens were obtained fromcadaveric multiorgan donors through the North Italian TransplantOrganization. Spleen cells were obtained by mechanical disruption of theorgan followed by density gradient centrifugation over Lymphoprep.

Differentiation of DC

CD14⁺ monocytes were isolated as the adherent fraction followingincubation for 1 hour in RPMI 1640 (Biowhittaker) supplemented with 10%FCS (Biowhittaker), 100 U/ml penicillin/streptomycin (Bristol-MyersSquibb), and 50 μM 2 mercaptoethanol (BioRad) (DC medium) at 37° C.Following extensive washing, adherent monocytes were cultured in 10ng/ml rhIL-4 (R&D Systems) and 100 ng/ml rhGM-CSF (R&D Systems) in DCmedium alone (obtained cells are named immature DC, iDC) or in thepresence of 10 ng/ml of rhIL-10 (BD, Bioscience, obtained cells arenamed Tr-DC) for 7 days. Alternatively, adherent monocytes were culturedin 10 ng/ml rhIL-4 (R&D Systems) and 100 ng/ml rhGM-CSF (R&D Systems) inDC medium alone for 5 days and matured with 1 μg/ml of LPS (SigmaAldrich, obtained cells are named mature DC, mDC) for additional 2 days.At day 7, immature DC (iDC), DC generated in the presence of IL-10(Tr-DC), and mature DC (mDC) were collected, irradiated (6000 RADS) andused to stimulate naïve CD4⁺ T cells or PBMC, therefore obtainingT(iDC), T(Tr-DC) and T(mDC) cell lines. The purity and maturation stateof DC were routinely checked by flow cytometric analysis to determineexpression of CD1a, CD14, CD83 and HLA-DR. In some experiments iDC,Tr-DC, and mDC were either left un-stimulated or activated with 50 ng/mlof rhIFN-γ (R&D Systems) and 200 ng/ml of LPS (Sigma) for additional 2days. In some experiments iDC, Tr-DC, and mDC were also tested forlevels of expression of CD11c, CD11b, CD71, CD80, CD83, CD86, ILT-2, (BDBiosciences), ILT-3 (Coulter Immunotech) and ILT-4 (kind gifts fromMarco Colonna), ICOS-L (eBioscience), and HLA-G (Exbion).

Purification of T Cells

CD4⁺ T cells were purified from PBMC by negative selection using theCD4⁺ T cell Isolation kit (Miltenyi Biotech), according to themanufacture's instructions. A portion of the resulting CD4⁺ T cells wascryopreserved for later use, and the remainders were depleted of CD45RO⁺cells using anti-CD45RO-coupled magnetic beads and LD negative selectioncolumns (Miltenyi Biotech). In the purified cells the proportion of CD4⁺CD45RO⁻CD45RA⁺ was consistently greater than 90%.

T Cell Differentiation Using DC.

1×10⁵ DC (iDC, Tr-DC, and mDC) were cultured with 1×10⁶ allogeneic naïveCD4⁺ CD45RO⁻ T cells in 1 ml of X-vivo 15 medium (Biowhittaker),supplemented with 5% pooled AB human serum (Biowhittaker), and 100 U/mlpenicillin/streptomycin (Bristol-Myers Squibb). After 6 or 7 days,rhIL-2 (40 U/ml) (Chiron) was added, and cells were expanded for anadditional 7 days. Fourteen days after initiation of the culture, Tcells were collected, washed and analyzed for their proliferativecapacity and cytokine secretion profile. In parallel, a proportion of Tcell lines was restimulated with immature, Tr-DC or mature DC from thesame allogeneic donor used in the primary culture. After 3 days, rhIL-2was added. One week after initiation of the second stimulation, T cellswere collected and analyzed for their proliferative capacity andcytokine secretion profile. Alternatively, 1×10⁵ DC (Tr-DC and mDC) werecultured with 1×10⁶ allogeneic PBMC cells in 1 ml. HLA-mismatched donorpairs, HLA-haploidentical pairs or HLA-matched un-related (MUD) pairswere tested. At day seven half of the medium, with or without cytokine,was replaced with fresh one. Ten days after initiation of the culture, Tcells were collected, washed and analyzed for their proliferativecapacity and cytokine secretion profile. Naïve CD4⁺ T cells or PBMCstimulated with immature DC are referred to as T(iDC) and thosestimulated with Tr-DC as T(Tr-DC) and those stimulated with mature DC asT(mDC). In some experiments, neutralizing anti-IL-10R (3F9, 30 mg/ml, BDPharmingen), anti-ILT-4 (10 μg/ml, kind gift from Marco Colonna) oranti-HLA-G (10 μg/ml 87G, Exbion) mAbs were added at the initiation ofeach round of stimulation and each time the cells were split. Cultureswith immature DC and Tr-DC typically resulted in 8-10-fold reduction inT-cell expansion compared to cultures stimulated with mature DC. Thisreduced recovery was not due to increased cell death as measured byannexinV staining (data not shown).

Alternatively, 5×10⁵ CD3-depleted PBMC were co-culture with the samenumber of allogeneic PBMC in a final volume of 1 ml, in the presence(CD3− APC+IL-10) or absence (CD3− APC) of exogenous IL-10 (10 ng/ml). Atday seven half of the medium, with or without cytokine, was replacedwith fresh one. At day ten cells were collected, washed, and analyzedfor their proliferative response in response of newly preparedCD3-depleted cells. PBMC stimulated with (CD3− APC) are referred asT(MLR) and with (CD3− APC+IL-10) as T(MLR/IL-10).

T Cell Differentiation Using L Cells.

Murine L cells transfectants expressing hCD32 (FCgRII), hCD58 (LFA-3),and hCD80 (48) were cultured in RPMI 1640 (Biowhittaker) supplementedwith 10% FCS (Biowhittaker), 100 U/ml penicillin/streptomycin(Bristol-Myers Squibb). L cells were detached by incubation withtrypsin-EDTA (Life-Technologies) and irradiated (700 rad) by x-raysource. Following washing, cells were plated in 24-well plates atinitial density of 4×10⁵ cells/ml in 500 μl volume of X-vivo 15 medium(Biowhittaker), supplemented with 5% pooled AB human serum(Biowhittaker), and 100 U/ml penicillin/streptomycin (Bristol-MyersSquibb), and 100 ng/ml of anti-CD3 (OKT3 Jansen-Cilag, Raritan, N.J.).After the L cells has adhered, 500 μl of naïve CD4⁺ T cells were addedat an initial density of 4×10⁵ cells/ml in complete medium.

All the experiments were conducted in the presence of recombinant humanIL-2 (100 U/ml) (Chiron) and human recombinant IL-15 (1 ng/ml) (R&D)(obtained cells are named Th0 cells). In addition, the following solublefactors were added as indicated: rhIL-10 (10 ng/ml) (BD, Bioscience),rh-IFN-α (5 ng/ml) (R&D) (obtained cells are named Tr1 cells), solubleHLA-G1 (30 ng/ml) alone (obtained cells are named Tg) or in combinationwith rhIL-10 (10 ng/ml) (obtained cells are named Tg10). T cells weresplited as necessary, IL-2 and IL-15 were replenished in all cultures.At day 7, T cells were collected, washed, counted, and restimulatedunder identical conditions for an additional 7 days. At day 14 of invitro culture, cells were collected, washed, counted, and analyzed fortheir profile of cytokine production and proliferative capacity. SolubleHLA-G1 was collected from culture supernatants of transfected line0.221-G (34).

Proliferation and Suppression of T Cells.

To analyze the proliferative capacity of T(iDC), T(Tr-DC), or T(mDC) inresponse to allogeneic APC, T cells were thawed and stimulated witheither allogeneic mDC (10:1, T:DC) or monocytes (CD3-depleted PBMCs,irradiated 6000 RADS) (1:1, T:monocytes) in a final volume of 200 μl ofmedium. To test for the capacity of T(iDC), T(Tr-DC), or T(mDC) cells tosuppress proliferation and/or cytokine production, autologous CD4⁺ Tcells were thawed and stimulated with allogeneic mDC (10:1, T:DC) in theabsence or in the presence of T(iDC), T(Tr-DC), or T(mDC) cells (1:1ratio) in a final volume of 200 μl of complete medium in 96 wellround-bottom plates. In some cultures, autologous CD4⁺ T cells werestimulated with allogeneic monocytes (CD3-depleted PBMCs, irradiated6000 RADS) (1:1, T:monocytes in the absence or in the presence ofT(Tr-DC) cells (1:1 ratio) and neutralizing anti-IL-10R (30 μg/ml, 3F9,BD Bioscience) and/or anti-TGF-β (50 μg/ml, 1D11, R&D systems) mAbs wereadded. After the indicated time, cells were either pulsed for 16 hourswith 1 μCi/well ³H-thymidine or supernatants were collected for analysisof IFN-γ production.

To test for the suppressive capacity of T cell lines via flow cytometry,naïve CD4⁺ T cells were labeled with CFSE (Molecular Probes) andstimulated with coated anti-CD3 mAb (10 μg/ml) and soluble anti-CD28 (1μg/ml) in the presence or absence (closed histograms) of Th0, Tg, Tg10,and Tr1 at 1:1 ratio. After 6 days, proliferation of the CFSE-labelednaïve T cells was determined by flow cytometric analysis.

Cytokine Determination: Intracytoplasmic Staining and ELISA.

To measure IFN-γ IL-2, IL-10, and TGF-β production, culture supernatantswere harvested 48, 72 and 96 hours after culture and levels of IFN-γwere determined by capture ELISA according to the manufacturer'sinstructions (BD Biosciences). To measure IL-10 and IL-12 produced byiDC, Tr-DC, and mDC, cells were left un-stimulated or activated with 50ng/ml of rhIFN-γ (R&D Systems) and 200 ng/ml of LPS (Sigma) foradditional 2 days. Supernatants were harvested after 48 hours. Levels ofIL-10 and IL-12 were determined by capture ELISA according to themanufacturer's instructions (BD Biosciences). The limits of detectionwere as follows: IFN-γ: 60 pg/ml; IL-10: 20 pg/ml; IL-12: 20 pg/ml.

Intracellular cytokines were detected by flow cytometry as previouslydescribed (47). Briefly, T cells (1×10⁶/ml) were stimulated withimmobilized anti-CD3 (1 μg/ml; OKT3, Jansen-Cilag, Raritan, N.J.) andTPA (10 ng/ml; Sigma) in complete medium. Prior to the culture, theplates were centrifuged for 5 min at 800×g. Three hours afteractivation, brefeldin A (10 μg/ml; Sigma) was added. Six hours afteractivation, T cells were collected, washed in PBS, and fixed with 2%formaldehyde. After fixation, T cells were permeabilized by incubationin PBS supplemented with 2% FCS and 0.5% saponin (Sigma). PermeabilizedT cells were incubated with anti-hIL-2, or anti-hIL-10, and FITC-coupledanti-hIFN-γ or anti-hIL-4 mAbs. All mAbs were obtained from PharMingen.After washing, cells were analyzed using a FACScan flow cytometer (BDBiosciences, Mountain View, Calif.), and data were analyzed withCellQuest software (BD Biosciences). Quadrant markers were setaccordingly to isotype-matched controls (data not shown).

Quantitative PCR.

Total RNA was extracted with Eurozol (Euroclone, Celbio), and cDNA wassynthesized using the high capacity cDNA archive kit (AppliedBiosystems). Levels of IL-10, IL-12 and HPRT mRNA were quantitated usingAssay on Demand real-time PCR kits (Applied Biosystems) with TaqManMaster Mix (Applied Biosystem). Samples were run in duplicate, andrelative expression of IL-10 and IL-12 was determined by normalizing toHPRT expression in each set of samples to calculate fold-change invalue.

FACS Analysis.

Anti-CD4, -CD25, -CD122, and -CD132, directly coupled with FITC and PEwere purchased from BD. Expression of IL-15Rα was determined withbiotinylated anti-IL-15Rα mAb (BD Bioscience) followed by streptavidinPE-conjugated (BD Bioscience). Expression of FOXP3 was determined byintracellular staining with FITC conjugated anti-FOXP3 mAb (clonePCH101, e-bioscience), following the manufacturer's instructions.Expression of CTLA-4, Granzyme A, and Granzyme B were determined byintracellular staining. Briefly, T cells were collected, washed in PBS,and fixed with 2% formaldehyde. After fixation, T cells werepermeabilized by incubation in PBS supplemented with 2% FCS and 0.5%saponin (Sigma). Permeabilized T cells were incubated with PE-labeledanti-CTLA-4 (BD Bioscience), anti-granzyme A (BD Bioscience), oranti-granzyme B (Caltag). After washing, cells were analyzed using aFACScan flow cytometer (BD Biosciences, Mountain View, Calif.), and datawere analyzed with CellQuest software (BD Biosciences).

Statistical Analysis.

All analysis for statistically significant differences were performedwith the student's paired t test. P values less than 0.05 wereconsidered significant. All cultures were performed in triplicate anderror bars represent the SD.

Results

IL-10 Prevents Down-Regulation of CD14 and Up-Regulation of CD1a on DC.

To determine the effect of exogenous IL-10 on the differentiation ofdendritic cells (DC), DC were differentiated from CD14⁺ monocytes in thepresence of IL-4 and GM-CSF for 7 days with exogenous IL-10 (Tr-DC),alternatively cells were differentiated with IL-4 and GM-CSF for 5 daysin the absence of IL-10 and then left unstimulated (immature DC, iDC) oractivated with LPS (mature DC, mDC) for additional 2 days. The authorsobserved that addition of exogenous IL-10 profoundly modified themorphology of the resulting cells. DC generated in the presence ofexogenous IL-10 (Tr-DC) were large, granular and displayed fewcytoplasmic expansions compared to immature and mature DC (FIG. 1A).IL-10 prevented the down-regulation of CD14 and the up-regulation ofCD1a as observed in immature and mature DC (FIG. 1B). Further phenotypiccharacterization of Tr-DC revealed an expression of CD83, CD80, and CD86similar to that observed in mature DC. CD11c, CD11b, and CD71 weresimilarly expressed by Tr-DC, immature, and mature DC (FIG. 1B).

The specific dendritic cell markers expressed by Tr-DC are summarized inTable I.

TABLE I Comparison of specific dendritic cell markers expressed byimmature, mature and Tr1 dendritic cells. tolerogenic cells (Tr-DC)Dendritic cell identified by the present type/specific markers iDC mDCmethod CD14 − − + CD11c + + + CD11b + + + CD83 − + + CD80 − + + CD86+/− + + CD71 + + + HLA-DR + + + CD1a + + −Tr-DC Secrete Higher Levels of IL-10 Compared to Immature DC.

Tr-DC secrete significantly higher amounts of IL-10 compared to iDC andmDC, whereas they secrete low amounts of IL-12, which are comparable tothose produced by iDC (FIG. 1C). Interestingly, upon activation with LPSand IFN-γ Tr-DC and iDC produce equal amounts of IL-10, but, in contrastto iDC, Tr-DC do not secrete significant levels of IL-12 (FIG. 1D).

These results were paralleled by the analysis of the mRNA levels of bothIL-10 and IL-12. IL-10 mRNA levels were significantly higher in Tr-DCcompared to iDC, whereas the mRNA levels for IL-12 were comparable inthe two cell types (FIG. 2A). Importantly, upon activation, Tr-DCdisplayed significantly higher amounts of mRNA for IL-10 compared toiDC. In addition, the mRNA level for IL-12 remains low (FIG. 2B).

These results indicated that Tr-DC are refractory to activation andmaintain their ability to express and secrete IL-10 but not IL-12. Alltogether these data clearly demonstrated that addition of exogenousIL-10 results in the differentiation of a novel subset of tolerogenic DC(Tr-DC), which are distinct from immature and mature DC.

Tr-DC Display Low Stimulatory Capacity.

Naïve CD4⁺ T cells stimulated with allogeneic Tr-DC display asignificantly lower proliferative response with a reduction inproliferation of 85±17% (mean±SD, n=24), when compared to naïve CD4⁺ Tcells primed with mDC (one representative experiment in FIG. 3A). Asexpected, iDC also poorly stimulated allogeneic naïve CD4⁺ T cells (amean±SD reduction in proliferation of 65±22% (mean±SD, n=24), comparedto proliferation induced by mDC). However, the stimulatory capacity ofTr-DC was significantly reduced compared to that of iDC (n=24,p=0.0008). Similarly, IFN-γ production by naïve CD4⁺ T cells stimulatedwith allogeneic Tr-DC was reduced when compared to production by naïveCD4⁺ T cells primed with mDC, and was significantly lower than thatinduced by iDC (a mean±SD reduction of 88±14% vs. 61±30% with Tr-DC andiDC, respectively, n=8, p=0.035) (one representative experiment in FIG.3B). Importantly, Tr-DC activated with IFN-γ and LPS maintained theirreduced stimulatory capacity as proliferation induced by activated Tr-DCwas significantly reduced compared to that generated by activated iDC (amean±SD reduction of 89±8% vs. 4±6% with Tr-DC and iDC, respectively,n=4, p<0.0001) (one representative experiment in FIG. 3C). Indeed,activated iDC acquired a mature phenotype and induced proliferation ofallogeneic CD4⁺ T cells similar to that of mDC (FIG. 3C). These datashow that upon activation Tr-DC maintain their low stimulatory capacity.

Tr-DC Induce T-Cell Anergy.

Tr-DC promote T-cell anergy, since naïve CD4⁺ T cells activated withTr-DC, become unable to proliferate when restimulated with mDC from thesame donor. After one round of stimulation, T cells generated withallogeneic Tr-DC [T(Tr-DC)] were already profoundly hypo-responsive tore-activation with mDC, whereas T cells stimulated with iDC [T(iDC)]were not. Reduction in Ag-induced proliferation of 82±14% and of 38±26%(mean±SD, n=8) was observed in T cells primed with Tr-DC and iDC,respectively, in comparison to T cells primed with mDC [T(mDC)] (onerepresentative donor in FIG. 4A). Similar results were obtained whenIFN-γ production by T cell lines re-challenged with mDC was measured(FIG. 3B). After two rounds of stimulation both T(iDC) and T(Tr-DC)cells were anergic and displayed an average reduction of proliferationof 72±12% (n=8) and 81±2% (n=8, not significant), respectively (FIG.4C). Thus, hypo-responsiveness could be acquired by naïve CD4⁺ T cellsstimulated with iDC only after repeated Ag stimulation (19, 20),conversely the authors show here that Tr-DC efficiently promote T-cellanergy following a single activation, indicating that Tr-DC are morepowerful inducers of T cell anergy compared to iDC.

Tr-DC Induce the Differentiation of IL-10-Producing Tr1 Cells.

T cells obtained after one round of stimulation with Tr-DC [T(Tr-DC)]contained a significant proportion of IL-10-producing cells (average:8%, range: 4-10%, n=9), and a low proportion of IL-4-producing cells(average: 4%, range: 0-8%). In these culture conditions, IL-2-producingcells were on average 7% (range: 2.3-12%), and IFN-γ-producing cellswere on average 16% (range: 3-20%). Conversely, T cells differentiatedwith iDC or mDC contained more IFN-γ-producing cells (on average 23%,range: 12-35% with iDC, and on average 40%, range: 22-67% with mDC) andlow IL-10-producing cells (on average 2.6%, range: 0.2-4.5% with iDC,and on average 2.7%, range: 0-5.2% with mDC). IL-10-producing cells weresignificantly high in T(Tr-DC) cell lines compared to T(iDC) and T(mDC)cell lines (p=0.00004) (FIG. 5A and FIG. 6).

After two rounds of stimulation with Tr-DC the proportion ofIL-10-producing cells increased (average: 12%, range: 9.7-13.2%, n=3)and the proportion of IL-2-producing cells (average: 3.6%, range:2.8-4.4%) and IFN-γ-producing cells (average: 15.4%, range: 11.2-21.3%)decreased (FIG. 5B). T cells induced by Tr-DC after one or two rounds ofstimulation, secrete similar amounts of TGF-β compared to cellsgenerated with immature DC [T(iDC)], and lower amounts compared to cellsgenerated with mature DC [T(mDC)] (FIG. 5C).

Phenotypic analysis of T(Tr-DC) cell lines revealed a percentage ofCD25⁺FOXP3⁺ cells similar to that observed in T(iDC) and T(mDC) celllines (FIG. 7A). The percentage of T cells expressing CD122, CD132,IL-15Rα and CTLA-4 was comparable among the T cell lines generated withdifferent DC, but T(Tr-DC) cells expressed higher levels of CTLA-4.Interestingly, the percentage of T(Tr-DC) cells expressing granzyme Bwas also higher compared to that observed in T(iDC) and T(mDC) cells,whereas the percentage of cells expressing granzyme A was comparableamong the three T cell populations (FIG. 7B). In summary, the T cellsinduced by Tr-DC are IL-10⁺⁺, TGF-β⁺, IL-4⁻ and IFN-γ and IL-2 negativeto low and are phenotypically similar to Tr1 cells.

Anergic T cells generated with Tr-DC suppress primary T-cell responses.Proliferation and IFN-γ production by naïve CD4⁺ T cells stimulated withmDC (MLR) was significantly suppressed by the addition of T(Tr-DC) cells(FIGS. 8A and B). Proliferation of naïve CD4⁺ T cells stimulated withmature DC had the kinetics of a primary response, peaking at day 4 ofculture (FIG. 8C), whereas, as previously demonstrated (20),proliferation of T(mDC) restimulated with mDC peaked at day 2 anddecrease at day 3 and 4, which is consistent with the kinetic of asecondary response (FIG. 8C). Interestingly, T(Tr-DC) cells acquiredsuppressive function after a single stimulation (FIGS. 8A and C),whereas addition of T(iDC) cells, obtained after one round ofstimulation, to the primary MLR resulted in increased proliferation atday 2 and 3 (FIGS. 8A and C). These data were mirrored when we examinedproduction of IFN-γ: addition of T(mDC) cells to the primary MLRresulted in an additive effect, whereas addition of T(Tr-DC) cellsresulted in an almost complete suppression of IFN-γ production (FIGS. 8Band D). As expected, after two rounds of stimulation T cell linesgenerated with iDC (T(iDC) and Tr-DC T(Tr-DC) suppress naïve primary MLR(FIG. 8C). These findings indicate that Tr-DC potently promote theinduction of anergic T cells with suppressive activity after singlepriming.

T cell lines generated with Tr-DC suppress primary MLR via an IL-10- andTGF-β mediated mechanism, since suppression was completely reversed bythe addition of neutralizing anti-IL-10R and anti-TGF-β mAbs (FIGS. 9Aand B). In addition, T(Tr-DC) cells do not require cell-cell contact fortheir suppressive activity since suppression of MLR was observed inexperiments performed using transwell chambers (data not shown).Furthermore, T(Tr-DC) cells were anergic towards allo-Ags but preservedtheir ability to proliferate in response to nominal Ags, such as TetanusToxoid and Candida Albicans (data not shown).

Overall, these data indicate that T cells generated by Tr-DC arefunctionally equivalent to Tr1 cells. Differentiation of Tr1 cells witheither iDC (20) or immuno-modulants, such as IL-10 alone (1) or incombination with IFN-α (47), or vitamin D3 and dexamethasone (49),requires repetitive Ag stimulations. Conversely, the authors show thatTr-DC promote the differentiation of IL-10-producing Tr1 cells after asingle stimulation. These findings are important for the prospectiveclinical application of Tr1 cells, since rapid and efficient ex-vivodifferentiation combined with Ag-specificity are desired characteristicsfor cellular therapy with regulatory T cells.

Tr-DC promote Tr1 cell differentiation via IL-10, since naïve CD4⁺ Tcells stimulated with Tr-DC in the presence of neutralizing anti-IL-10Rwere not anergic (data not shown) and did not acquire suppressiveactivity (FIGS. 9C and D). These results indicate that IL-10 is requiredfor the differentiation of Tr1 cells by Tr-DC and are in line with ourprevious study demonstrating that autocrine production of low amounts ofIL-10 by iDC is a necessary component for induction of Tr1 cells afterrepetitive Ag stimulation (20).

Differentiation of Tr1 Cells by Tr-DC Requires ILT-4/HLA-G Pathway.

Tr-DC express significantly higher levels of immunoglobulinlike-transcript (ILT)-2, ILT-3, ILT-4, and HLA-G, compared to iDC (FIGS.10A and B). Interestingly, no differences in the expression of ICOS-Lwere observed between Tr-DC and iDC (FIGS. 10A and B). Severalimmuno-modulants such as IL-10 (29), IFN-α (50), and vitamin D3 (51)have been reported to up-regulate ILT-3 and ILT-4 expression on DC.Interestingly, the same compounds have been shown to promotedifferentiation of regulatory T cells (47, 49). Furthermore, DCexpressing ILT-4 or HLA-G are poor stimulators and promote the inductionof anergic CD4⁺ T cells (43, 50). It has been recently described thatIL-10 inhibits endothelium-dependent T-cell activation by promotingILT-4 expression on endothelial cells (52).

ILT-4 expressed on Tr-DC plays a role in the induction of Tr1 cells,since stimulation of naïve CD4⁺ T cells with Tr-DC in the presence ofneutralizing anti-ILT-4 mAb prevented the induction of anergic T cells(FIG. 10C) with suppressive activity (FIG. 10D, and FIG. 11A). SinceILT-4 binds to HLA-G, a non-classical HLA class I molecule (44), theauthors next investigated the expression of HLA-G on CD4⁺ T cellsactivated with Tr-DC. Freshly isolated naïve CD4⁺ T cells express meanpercentage of HLA-G of 2.2±0.9% (mean±SD, n=4) (data not shown) butafter priming with Tr-DC the expression was up-regulated and T(Tr-DC)cell lines expressed significantly higher levels of HLA-G compared to Tcells stimulated with iDC, and with mDC (FIG. 10E). In T-cell culturesstimulated with Tr-DC the percentage of CD4⁺HLA-G⁺ T cells was 17.7±6.7%versus 6.7±3.4% CD4⁺HLA-G⁺ T cells in cultures with iDC (n=7, p=0.0014),and 11.1±7.7% CD4⁺HLA-G⁺ T cells in cultures with mDC (n=7, p=0.05).Importantly, the induction of HLA-G expression on T cells cultured withDC-10 was IL-10 dependent since priming of naïve CD4⁺ T cells with Tr-DCin the presence of anti-IL-10R blocking mAb prevented HLA-Gup-regulation (FIG. 10E). These results indicate that autocrineproduction of IL-10 by Tr-DC not only up-regulates ILT-4 and HLA-G on DC(data not shown), but it is also required for HLA-G up-regulation onCD4⁺ T cells. The role of IL-10 in promoting HLA-G expression on APC hasbeen previously shown (53), but this is the first demonstration thatIL-10 up-regulates HLA-G expression also on CD4⁺ T cells. To furtherprove that ILT-4/HLA-G interaction leads to Tr1 cell differentiationdriven by Tr-DC, naive CD4⁺ T cells were stimulated with Tr-DC in thepresence of neutralizing anti-HLA-G mAb. Activation of T cells withTr-DC in the presence of neutralizing anti-HLA-G mAb prevented theinduction of anergic T cells (FIG. 10F) with suppressive activity (FIG.10G, and FIG. 11B). Taken together these results demonstrate thatinteraction between ILT-4 and IL-10-induced HLA-G is required for Tr1cell differentiation. Moreover, these data also suggest that theindispensable role of IL-10 in Tr1 cell induction is due to its abilityto up-regulate the tolerogenic molecules ILT-4 and HLA-G on both DC andT cells. HLA-G is a potent immuno-modulant, and is the major player inmaintaining foetal-maternal tolerance (31). HLA-G inhibits cytolyticactivity of NK and CTL (36), and allo-specific T-cell proliferation(38). Interestingly, a positive correlation between allograft acceptanceand HLA-G expression on both graft cells (39, 40) and T cells (38) hasbeen described, supporting a role of HLA-G in modulating allo-responses.Furthermore, it has been previously reported that HLA-G modulates DCfunction (54). Engagement of ILT-4 on DC by soluble HLA-G prevents theup-regulation of costimulatory molecules and inhibits their maturation(45), and DC treated with soluble HLA-G promote the induction ofanergic/suppressor CD4⁺ T cells (46). Here the authors demonstrate a keyrole of membrane-bound HLA-G in inducing human adaptive regulatory Tcells.

In the present model system, IL-10 produced by tolerogenic Tr-DCinhibits T-cell proliferation and cytokine production, promotes T-cellanergy, up-regulates expression of ILT-4 on DC and modulates theexpression of HLA-G on DC and T cells. ILT-4/HLA-G interaction enhancesIL-10 production by Tr-DC amplifying this “tolerogenic” loop. Moreover,signals through HLA-G on T cells might contribute to T-cell anergyinduction by inhibiting T-cell activation. It has been indeed recentlyproposed that HLA-G can act as signalling molecule (55). It cannot beexcluded that additional pathways might synergize with the ILT-4/HLA-Ginteraction in promoting Tr1 cell differentiation. Tr-DC express ILT-2,the second ligand of HLA-G, and ILT-3, which might co-operate withILT-4/HLA-G in inducing a tolerogenic response. Moreover, Tr-DC expressHLA-G that might promote T-cell anergy by interacting with ILT-2 on Tcells. It has been reported that ILT-2 engagement on T cells inhibitsTCR-mediated signalling and prevents T-cell proliferation (56, 57).

Tr-DC Induce Anergic T Cells in Haplo-Identical and HLA-MatchedUn-Related Pairs.

The authors next determine the ability of Tr-DC to induce T-cell anergyin pairs with different HLA disparities. Similarly to that observedusing naïve CD4⁺ T cells as responder cells, they demonstrated thatTr-DC elicited a lower proliferative response by allogeneic PBMC, withan average reduction of 89±10% (n=17, p<0.0005) and IFN-γ productionwith an average reduction of 95±10% (n=17, p<0.05), compared to thatelicited by mature DC (FIG. 12). They then determined the ability ofTr-DC to induce anergic T cells. PBMC were co-cultured with Tr-DC at a10:1 ratio for ten days as described in the Material and Methods, andsubsequently tested for their ability to proliferate in response to theoriginal allogeneic mature DC. PBMC primed with Tr-DC [T(Tr-DC)] werehypo-responsive to re-activation with the original allogeneic mature DC,whereas PBMC primed with mature DC [T(mDC)] were highly proliferative,as expected. An average reduction of 80±8% (n=18, p<0.0005) inAg-induced proliferation of cells generated with Tr-DC was observed incomparison to PBMC primed with mature DC (FIG. 13).

The authors then determined the ability of Tr-DC to induce anergic Tcells in haplo-identical and HLA-matched un-related (MUD) pairs. Resultsclearly demonstrated that Tr-DC induced anergic T cells in bothsettings. In FIG. 14 inhibition of secondary responses inhaplo-identical pairs tested is shown. The mean value of anergy inducedby Tr-DC was 78±8% (n=4, p=0.0007). Moreover, Tr-DC induced anergic Tcells in MUD context with an average of 78±14% (n=3, p<0.0005) (FIG.15). All together these results clearly indicate that cells generatedwith IL-10 are potent tolerogenic DC that induce anergic T cells,containing precursors or already differentiated Tr1 cells, to be used ascellular therapy to prevent/cure GvHD and organ graft rejection.

Comparison Between the Protocol to Anergize Cells with Exogenous IL-10and with Tr-DC.

The ability to induce anergic T cells in haplo-identical pairs usingTr-DC was compared to that obtained using exogenous IL-10 and CD3⁻APC.PBMC were co-cultured with either Tr-DC or mDC at a 10:1 ratio or withCD3-depleted cells in the absence or presence of exogenous IL-10 at aratio 1:1 for ten days, as described in the Material and Methods, andsubsequently tested for their ability to proliferate in response to theoriginal allogeneic mature DC. PBMC primed with both Tr-DC T(Tr-DC)] andCD3-depleted cells+IL-10 [T(MLR/IL-10)] were hypo-responsive tore-activation with mature DC. An average reduction of 78±8% (n=4), andof 67±33% (n=4) in Ag-induced proliferation of cells generated withTr-DC and monocytes+IL-10 (MLR/IL-10) respectively, in comparison toPBMC primed with mature DC, was observed (FIG. 16). Importantly, whilethe protocol to anergize T cells with IL-10 fails to be successful ininducing high anergy in all of the donors, high anergy is obtained in Tcell from all individuals tested with Tr-DC (Table II).

TABLE II Tr-DC induce anergic T cells in all haplo-identical pairs. 1 23 4 ANERGY % Tr1-DC 79 79 68 88 CD3⁻APC + IL-10 82 99 35 36

PBMC were stimulated with Tr-DC at 10:1 ratio or with CD3-depletedcells+IL-10 at 1:1 ratio for ten days. T-cell lines were tested fortheir ability to proliferate in response to mature allogeneic DC.Proliferative responses were evaluated by thymidine incorporation after48 h of culture. Numbers represent the % of anergy compared to mDC. 1,2, 3 and 4 represent different donors.

Generation of Tr-DC for Clinical Use.

To generate anergized T cells for clinical use the authors optimized thecondition for the differentiation of Tr-DC. To this end theydifferentiated Tr-DC using medium containing either FCS or human serum(HS). The results obtained in eight different donors indicate that thephenotype of the differentiated Tr-DC in medium containing human serumis comparable to that obtained Tr-DC differentiated in medium containingFBS (FIG. 17A). Moreover, Tr-DC generated in medium containing FBS or HSare comparable in the ability to induce anergic T cells. An averagereduction of 88±14% (n=2) and 78±8% (n=2) in Ag-induced proliferation ofT cells primed with Tr-DC [T(Tr-DC)] differentiated in HS and in FBScells, respectively, in comparison to T cells primed with mature DC[T(mDC)] was observed (FIG. 17B). Collectively, these data indicate thatTr-DC differentiated in medium containing HS are phenotypicallyidentical to Tr-DC generated in medium containing FBS, and areequivalent in inducing anergic T cells.

Scale Up Procedure to Differentiate Tr-DC for Clinical Use.

To establish a procedure to generate Tr-DC for clinical use the authorsdifferentiated Tr-DC in flask, and their phenotype and biologicalfunctions were compared to those of cells generated in plate. Tr-DCgenerated in flask and in plate are equivalent in term of phenotype andinduce anergy in responder T cells in comparable manner. An averageinhibition of 81±8% (n=4) and 78±4% (n=4) in Ag-induced proliferation ofT cells primed with Tr-DC [T(Tr-DC)] generated in plate and in flask,respectively, in comparison to T cells primed with mature DC [T(mDC)],was observed (FIG. 18 and data not shown). These results indicate thatTr-DC generated in flask are comparable to that obtained in plate ininducing anergic T cells, and therefore suitable for clinicalapplication according to the following scheme.

A proposed cell therapy protocol is illustrated by Scheme 1, shown byFIG. 25.

Anergic T cells can be injected by systemic route with a concentrationranging between 10² to 10⁸ CD3+ cells/kg of body weight.

Tr-DC Also Exist In Vivo

It should be noted that Tr-DC (CD11c⁺CD11b⁺CD14⁺CD83⁺CD1a⁻) wereidentified in peripheral blood of normal donors where they represent3.2±2.2% (mean±SD, n=6) of the mononuclear cells (FIG. 19A). Todetermine whether Tr-DC are present also in secondary lymphoid organs,the authors analyzed the spleen from normal donors. Interestingly, usingCD14, CD11b, CD11c, CD83 and CD1a as markers, they demonstrated thatTr-DC cells are present in human spleen and represent 6.2±1.6% (mean±SD,n=4) of the total cells (FIG. 19B). Importantly, ILT-2, ILT-3, ILT-4,and HLA-G were also highly expressed on Tr-DC present in peripheralblood and spleen (FIGS. 20A and 20B). These findings show that Tr-DC area distinct DC subset, which is not only inducible in vitro in thepresence of exogenous IL-10, but also exist in vivo and thus can bedirectly isolated from the subject samples such as blood, spleen orlymph nodes.

Taken together these data indicate that Tr-DC, which areILT3⁺ILT4⁺HLA-G⁺IL-10⁺⁺IL-12^(low/neg), represent a distinct subset oftolerogenic cells in vivo and can be differentiated in vitro withexogenous IL-10. Tr-DC produce high levels of IL-10 and are powerfulinducers of Tr1 cells. Tr-DC drive Tr1 cell differentiation via theIL-10-dependent ILT-4/HLA-G pathway, since blocking of these tolerogenicmolecules prevents Tr1 cell induction. Tr-DC set the stage for inductionof regulatory T cells by secreting IL-10 that inhibits T-cellproliferation, up-regulates ILT-2, ILT-3, ILT-4, and HLA-G on DC, andinduces HLA-G on T cells. The interaction between HLA-G and ILT-4enhances IL-10 production by DC-10, which consequently may promote denovo expression of ILT-2, ILT-3, ILT-4, and HLA-G on other immature DC(FIG. 21). IL-10-induced HLA-G on DC and T cells represents a crucialcomponent of the ILT-4 mediated mechanism of Tr1 cell differentiation.Thus, DC expressing ILT-4 and HLA-G and producing IL-10 are tolerogenicDC, which may be induced in vivo by antigens and pathogens as a way toescape immune responses. Overall our data demonstrate the central roleof Tr-DC in the differentiation of adaptive Tr1 cells and identify ILT-4and HLA-G as key surface molecules for tolerance induction.

Soluble HLA-G Induces Regulatory T Cells.

The authors next investigated the role of soluble HLA-G1 (sHLA-G) (TableIII) in promoting regulatory T cells differentiation using a system ofartificial APC consisting in murine L-cells co-transfected with hCD32,hCD80, and hCD58 (48).

TABLE III Sequence of soluble HLA-G1 (SEQ ID No. 1)MVVMAPRTLFLLLSGALTLTETWAGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGYYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSK EGDGGIMSVRESRSLSEDL

In this model repetitive stimulation of naïve human CD4⁺ T cells in thepresence of IL-10 and IFN-α polarized T cells into IL-10-producing Tr1cells with low proliferative capacity and suppressor functions (47). Theauthors investigated whether IFN-α can be substituted by sHLA-G in thissetting to promote Tr1 cell differentiation. Repetitive stimulation ofnaïve CD4⁺ T cells with anti-CD3 cross-linked on CD32⁺CD58⁺CD80⁺ L cellsin the presence of sHLA-G alone (Tg) or in combination with IL-10 (Tg10)induce the differentiation of a population of CD4⁺ T cells that produceTGF-β, intermediate levels of IL-10, low amounts of IFN-γ, but no IL-2,and IL-4 (FIGS. 22A and B). Tg cells obtained after two rounds ofstimulation with anti-CD3 cross-linked on CD32⁺CD58⁺CD80⁺ L cells in thepresence of sHLA-G contained an intermediate proportion ofIL-10-producing cells (average: 4%, range: 2-9%, n=5), and anintermediate proportion of IL-4-producing cells (average: 7%, range:2-11%). In these culture conditions, IL-2-producing cells were onaverage 7% (range 4.2-9.9%), and IFN-γ-producing cells were on average14% (range: 6-23%). Tg10 cells obtained after two rounds of stimulationwith anti-CD3 cross-linked on CD32⁺CD58⁺CD80⁺ L cells in the presence ofsHLA-G and IL-10 contained an intermediate proportion of IL-10-producingcells (average: 5%, range: 2-9%, n=5), and an intermediate proportion ofIL-4-producing cells (average: 7%, range: 3-11%). In these cultureconditions, IL-2-producing cells were on average 6% (range 3-8%), andIFN-γ-producing cells were on average 12% (range: 4-22%). Conversely, Tcells differentiated with anti-CD3 cross-linked on CD32⁺CD58⁺CD80⁺ Lcells in the presence of IL-10 and IFN-α (Tr1) contained a higherproportion of IL-10-producing cells (on average 12%, range: 5-16%, n=7),IFN-γ-producing cells (on average 31%, range: 18-42%, n=7), andIL-2-producing cells (on average 13%, range: 6-20%, n=7), but lowIL-4-producing cells (on average 3%, range: 1.3-7%, n=7), (FIG. 23A).These results were paralleled with resulted obtained by measuringcytokine in culture supernatants (FIG. 22B).

Phenotypic analysis of Tg and Tg10 cell lines revealed a percentage ofCD25⁺FOXP3⁺ cells similar to that observed in Tr1 cell lines (FIG. 23A).The percentage of Tg and Tg10 cells expressing CTLA-4 was highercompared to that observed in Tr1 cells (FIG. 23B). Interestingly, thepercentage of T cells expressing HLA-G was comparable among the T celllines (Tg, Tg10, and Tr1 cells), but higher compared to that of Th0cells. Interestingly, the percentage of Tg, Tg10 and Tr1 cellsexpressing granzyme B was also higher compared to that observed in Th0cells, whereas the percentage of cells expressing granzyme A wascomparable among the Tg and Tg10 but lower compared to Tr1 cells (FIG.23C). In summary, the T cells differentiated with anti-CD3 cross-linkedon CD32⁺CD58⁺CD80⁺ L cells in the presence of sHLA-G alone (Tg) or incombination with IL-10 (Tg10) are phenotypically similar to Tr1 cellsbut secrete lower amount of IL-10 and IFN-γ and do not secrete IL-2.

T cells differentiated with anti-CD3 cross-linked on CD32⁺CD58⁺CD80⁺ Lcells in the presence of sHLA-G alone (Tg) or in combination with IL-10(Tg10) display low proliferative capacity (data not shown) and suppressprimary T-cell responses. Proliferation of naïve CD4⁺ T cells stimulatedwith coated anti-CD3 and soluble anti-CD28 mAbs was significantlysuppressed by the addition of Tg and Tg10 cells (FIG. 24A).

In summary, the present invention indicates that:

-   -   i) IL-10 modulated DC (Tr-DC) are a novel subset of tolerogenic        DC that are CD14⁺CD11c⁺CD11b⁺CD83⁺HLA-DR⁺CD1a⁻,    -   ii) IL-10 modulated DC (Tr-DC) are a novel subset of tolerogenic        DC that display a mature myeloid phenotype (CD80⁺CD86⁺)    -   iii) IL-10 modulated DC (Tr-DC) are a novel subset of        tolerogenic DC that express immunoglobulin-like transcript        (ILT)-2, ILT-3, ILT-4, and HLA-G    -   iv) IL-10 modulated DC (Tr-DC) are a novel subset of tolerogenic        DC that secrete high levels of IL-10 and low levels of IL-12,        and are refractory to activation and maturation in vitro.    -   v) Tr-DC induce anergic T cells.    -   vi) Anergic T cells induced by Tr-DC are regulatory T cells        phenotypically and functional similar to Tr1 cells.    -   vii) Tr-DC induce anergic T cells in pairs with different HLA        disparities, which can be used as cellular therapy to prevent        GvHD and organ allograft rejection.    -   viii) Soluble HLA-G1 alone or in combination with IL-10 promotes        the differentiation of a population of CD4⁺ T cells with        suppressive activity.

In addition, anergized T cells generated with Tr-DC:

-   -   contain a significant proportion of Tr1 cells    -   are stable    -   are antigen-specific    -   are able to suppress Ag-specific primary responses    -   are induced by shot-term culture.

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The invention claimed is:
 1. An in vitro method for generating apopulation of T(Tr-DC) cells having regulatory activity, the methodcomprising the steps of: a) generating a tolerogenic human dendriticcell population (Tr-DC) population from a subject that comprises themarker phenotype: CD14⁺, CD11c⁺, CD11b⁺, CD1a⁻, CD83⁺, CD80⁺CD86⁺,HLA-DR⁺, and CD71⁺, wherein at least about 92% of the generated humanTr-DC are CD86⁺ positive; b) irradiating the Tr-DC population of a); c)isolating peripheral blood mononuclear cells (PBMCs) or CD4⁺ T cellsfrom a subject and d) stimulating said isolated PBMCs or the CD4⁺ Tcells, in culture, with an effective amount of said irradiated Tr-DCcell population, to generate a population of T(Tr-DC) cells; wherein thesubject of step a) is the same or different from the subject of step c).2. The method of claim 1, wherein the irradiation is conducted with anexposure of 6000 Rads from an X-ray source.
 3. The method of claim 1,wherein the population of Tr-DC is obtained from a subject by a processcomprising the steps of: a) collecting a sample from the subject; b)generating Tr-DC cells from the collected sample that express: CD14⁺,CD11c⁺, CD11b⁺, CD1a⁻, CD83⁺, CD80⁺, CD86⁺, HLA-DR⁺, and CD71⁺, whereinat least about 92% of the generated human Tr-DC are CD86⁺ positive andwherein the sample is a blood sample, a spleen sample or a lymph nodesample.
 4. The method of claim 3, where the step of generating Tr-DCcells comprises the steps of: a) collecting peripheral blood mononuclearcells (PBMCs) from a subject; b) isolating adherent cells from collectedPBMCs; and c) exposing said isolated adherent cells at the start ofculture on day 0 to an effective amount of GM-CSF, IL-4 and IL-10 togenerate the Tr-DC cells.
 5. The method of claim 1, wherein the ratio ofPBMC or CD4⁺ T cells to irradiated Tr-Dc is 1:10.
 6. The method of claim1, wherein the isolated PBMCs or isolated CD4+ T cells are stimulatedwith an effective amount of the irradiated Tr-DC cell population for 10days.
 7. The method of claim 1, wherein the isolated PBMCs arestimulated with an effective amount of the irradiated Tr-DC cellpopulation in the presence of IL-10.
 8. The method of claim 1, whereinthe Tr-DC, and the PBMCs or the CD4⁺ T cells originate from differentsubjects.
 9. The method of claim 1, wherein the generated population ofT(Tr-DC) cells is anergic; and comprises cells with the following markerphenotype: IL-10⁺⁺, TGF-β⁺, IL-4⁻, IFN-γ⁺ and IL-2 negative to low. 10.A method of inducing or restoring immune tolerance in a subject in needthereof comprising administering to the subject in need thereof thepopulation of T(Tr-DC) cells produced by the method of claim
 1. 11. Themethod of claim 10, wherein the population of T(Tr-DC) cells is anergic;and comprises cells with the following marker phenotype: IL-10⁺⁺,TGF-β⁺, IL-4⁻, IFN-γ⁺ and IL-2 negative to low.
 12. A method fortreating a disease in a subject, wherein the disease is selected fromthe group consisting of graft versus host disease, organ graftrejection, autoimmune disease, allergies, asthma, and chronicinflammatory disease in a subject in need thereof, the method comprisingadministering to the subject the population of T(Tr-DC) cells producedby the method of claim
 1. 13. The method of claim 12, where theautoimmune disease is selected from the group consisting of type 1diabetes mellitus, autoimmune entheropathy, rheumatoid arthritis,systemic lupus erythematosus, multiple sclerosis and psoriasis.
 14. Themethod of claim 12, where the chronic inflammatory disease is selectedfrom the group consisting of inflammatory bowel disease, Crohn's diseaseand vasculitis.
 15. The method of claim 12, wherein the allergy isatopic dermatitis.
 16. The method of treating a disease according toclaim 12, wherein the disease is graft versus host disease or organgraft rejection.
 17. The method of claim 12, wherein the population ofT(Tr-DC) cells is anergic; and comprises cells with the following markerphenotype: IL-10⁺⁺, TGF-β⁺, IL-4⁻, IFN-γ⁺ and IL-2 negative to low. 18.A method for treating a disease in a subject, wherein the disease isselected from the group consisting of: immune dysfunction,Polyendocrinopathy Enteropathy X-linked (IPEX) syndrome, AutoimmunePolyendocrinopathy-Candidiasis-Ectodermal Dystrophy (APECED) syndrome,and OMENN's syndrome, the method comprising administering to the subjectthe population of T(Tr-DC) cells produced by the method of claim
 1. 19.The method of claim 18, wherein the population of T(Tr-DC) cells isanergic; and comprises cells with the following marker phenotype:IL-10⁺⁺, TGF-β⁺, IL-4⁻, IFN-γ⁺ and IL-2 negative to low.